Epithelial cells play critical roles in many organs and are responsible for elemental processes like nutrient uptake or waste product secretion. There is a fundamental gap in understanding how epithelial cells maintain their intrinsic cell polarity throughout their lifetime, both during normal activity and during wound healing processes. To maintain cell polarity, epithelial cells must continuously sort newly synthesized and internalized transmembrane proteins to the correct membrane domains (basolateral or apical). Essential to this process in most epithelia is the cytosolic adaptor complex AP-1B that delivers proteins to the basolateral membrane in transport vesicles. How AP-1B vesicles form and how vesicle formation is regulated are unresolved questions. The long-term goals are to understand how AP-1B is regulated on a molecular level and how deregulation of AP-1B may contribute to disease development. The central hypotheses are that a network controlled by AP-1B during constitutive basolateral sorting may also be used by AP-1B to control epithelial cell migration. Furthermore, we hypothesize that only a few critical amino acids in AP-1B are necessary to confer specificity to these processes. The rationale behind these hypotheses is that most of the proteins that we discovered to work with AP-1B in basolateral sorting are also known for driving cell migration and cancerous transformations. Moreover, while AP-1B controls this cell migration network during basolateral sorting, the closely related AP-1A does not. Guided by strong preliminary data, these hypotheses will be tested by pursuing two specific aims: 1) How does AP-1B facilitate exocyst recruitment during vesicle formation; and 2) How does AP-1B control cell migration. Under the first aim, we will use site-directed mutagenesis to determine critical amino acid residues in AP- 1B to unravel how AP-1B controls basolateral sorting. Mutant complexes will be analyzed using established protocols in combination with state-of-the-art confocal and electron microscopy. Under the second aim, we will examine a function for AP-1B in cell migration using state-of-the-art imaging techniques including live-cell imaging and super-resolution immunofluorescence microscopy in combination with depletion of AP-1B and other candidate proteins using gene knock down approaches. The proposal is innovative, because it is the first one to analyze AP-1B in cell migration, which will potentially broaden its functions i epithelial cell homeostasis from basolateral sorting in fully polarized cells to cell migration aftr wounding. The proposed research is significant, because it will result in novel insights regarding the contribution of AP-1B function in health and disease, and will lead to an advanced understanding of AP-1B regulation during constitutive basolateral sorting and epithelial cell migration during normal wound healing and in diseases. This has the potential to inform the development of new drug screens toward novel target proteins and future translational research.